Analysis of the association between serum antiaging humoral factor klotho and cardiovascular disease potential risk factor apolipoprotein B in general population

Cardiovascular disease (CVD) is a prevalent health issue, and various risk factors contribute to its development, including blood lipids, blood pressure, diabetes, smoking, and alcohol consumption. Apolipoprotein B (ApoB) is related to CVD. ApoB is present on the surface of low-density lipoprotein (LDL), and its cellular recognition and LDL uptake are mainly achieved through recognition. It plays a crucial role in the diagnosis and treatment of CVD. This study aims to investigate the relationship between Klotho and ApoB in the general population of the United States as the correlation between serum Klotho and apoB is currently unknown. These findings could potentially guide the development of future treatments for CVD. This study utilized data from the National Health and Nutrition Examination Survey (NHANES) collected between 2007 and 2016. A linear regression model and smooth curve fitting were conducted to analyze the relationship between serum Klotho and apoB. The results indicate a negative correlation between serum Klotho concentration and apoB concentration (β = −71.7; 95% confidence interval [CI]: −120.8, −22.6; P = .005). After adjusting for confounding variables, the negative correlation between apoB concentration and serum Klotho concentration became more significant (β = −91.8; 95% CI: −151.3, −32.2; P = .004). When apoB concentration was converted from a continuous variable to a categorical variable (tertiles: T1 <0.8 g/L; T2: ≥0.8 g/L to <1.0 g/L; T3: ≥1.0 g/L), the serum klotho level of participants in the highest tertile (≥1.0 g/L) was −44.8 pg/mL (95% CI: −86.3, −3.2; P = .040) lower than that in the lowest tertile (<0.8 g/L). The smooth curve fitting diagram revealed differences in the relationship between serum Klotho concentration and apoB among individuals with different CVD risk factors. This study demonstrates a significant negative correlation between serum Klotho concentration and apoB concentration, even after controlling for confounding factors. The findings suggest that serum Klotho and apoB may be involved in the development of CVD, and targeting these factors could be a potential approach for CVD prevention and treatment.


Introduction
Klotho is a kidney protective factor that has been extensively studied. The human Klotho gene, located on chromosome 13, encodes Ι Type single transmembrane glycoprotein membrane binding Klotho. Its relative molecular weight is 130 kDa, and it is composed of 1012 amino acids. [1] Membrane-bound Klotho passes through α and β Cut to form a truncated Medicine α-Klotho protein, also known as soluble α-Klotho protein. [2] The Klotho protein discussed in this paper is α-Klotho, which is widely expressed in various human tissues, with the kidney having the highest level of Klotho gene and protein expression. [3] Specifically, Klotho is mainly expressed in the distal convoluted tubules of the kidney and only a small amount in the proximal convoluted tubules. [4] Identified in 1997 as an anti-aging gene, Klotho is also closely related to kidney function and acts as a renal protective agent. [5][6][7][8] Additionally, Klotho can serve as a marker of chronic kidney disease (CKD), as the level of Klotho in the kidney of CKD patients is significantly lower than that in a regular control group. [9] In experiments with rats with diabetic nephropathy, azithromycin nephropathy, or spontaneous hypertension, supplementation of Klotho was found to reduce podocyte and glomerular hypertrophy, indicating its protective role in the kidney. [10][11][12] The relationship between serum Klotho and cardiovascular diseases (CVD) is intricate. On the one hand, the Klotho protein is closely linked to CKD, [9] and on the other hand, some association of Klotho with CVD was found in CKD patients. Studies have revealed that FGF23 and Klotho have a common effect in CKD patients, regulating mineral metabolism and participating in the occurrence and death of CVD. [13] Six et al [14] found that elevated Klotho and FGF23 levels could induce the contraction of aortic rings and the production of oxidative stress substances in vascular smooth muscle cells. At the same time, Klotho protein can partially reverse the vasoconstriction caused by FGF23, promote aortic relaxation (both may be achieved by increasing NO secretion), and increase the production of NO in human vein endothelial cells. Therefore, Klotho can reduce vasoconstriction and protect blood vessels to some extent.
Previously, evaluating the concentration of serum low-density lipoprotein cholesterol (LDL-C) was a critical factor in managing the risk of atherosclerotic cardiovascular disease (ASCVD). [15,16] However, recent evidence suggests that while lipid-lowering therapy targeting serum LDL-C reduces the risk of ASCVD in the population, some individuals with normal or low serum LDL-C concentrations still experience ASCVD-related events, and some individuals experience atherosclerosis progression. [17] This indicates that it is not the best strategy for all patients to manage ASCVD only by focusing on serum LDL-C concentration and that attention should also be paid to the impact of apolipoprotein B (ApoB). ApoB is a granular apolipoprotein that can be found in a variety of lipoproteins. Each lipoprotein contains 1 molecule of apoB, which is surrounded by a single layer of phospholipids. [18] ApoB provides structural stability to the lipoprotein particles and remains unchanged throughout the metabolic process. The core of the lipoprotein particles consists of varying amounts of triglycerides and cholesterol. [19] ApoB acts as a carrier and is present in different types of lipoproteins, including LDL, intermediate-density lipoprotein, very low-density lipoprotein (VLDL), chyle granules, and LP(a). ApoB100 is mainly found in LDL, while the rest is found in VLDL, intermediate-density lipoprotein, and LP(a), and apoB48 can be found in chyle granules. As each lipoprotein particle contains 1 molecule of apoB, the plasma apoB is equal to the total number of apoB48, apoB100, and LP(a) particles. [20] Since apoB48 is rare, the clinical measurement of apoB equals the concentration of apoB100. The total amount of apoB is the sum of VLDL, LDL, and LP(a) particles, with LDL accounting for the highest amount. This makes apoB highly correlated with LDL. As elevated apoB may predict CVD risk in individuals with normal or low LDL-C levels, exploring the relationship between apoB and Klotho is beneficial for screening potential CVD patients in this population. Currently, some guidelines suggest using apoB as a secondary intervention target of blood lipid management to reduce the residual risk of ASCVD in patients. [21,22] Therefore, apoB plays an essential role in the diagnosis and treatment of CVD.
Based on the contextual information, it is plausible to suggest a potential correlation between serum Klotho and apoB, which requires further exploration. Nevertheless, the existing research on the relationship between serum Klotho and apoB is limited. Therefore, this study aims to investigate the association between serum Klotho and apoB, using data collected from the National Health and Nutrition Examination Survey (NHANES) conducted between 2007 and 2016.

Study design and population
The NHANES is a comprehensive health and nutrition survey conducted by the National Center for Health Statistics in the United States. Since its inception in the early 1960s, NHANES has aimed to assess the health and nutritional status of individuals across the United States. This survey utilizes family interviews and physical examinations to gather detailed biological, social, psychological, behavioral, and demographic information at no cost to participants. In this cross-sectional study, we utilized NHANES data from 2007 to 2016 to investigate the relationship between apoB and serum Klotho in the general population.

Sample selection
We analyzed data from the NHANES database, which included 50,588 participants from the years 2007 to 2016. The analysis included demographic data such as age, gender, race, ratio of family income to poverty, and education level. Additionally, we looked at CVD risk factors, including hypertension, high cholesterol levels, diabetes, body mass index (BMI), drinking, and smoking variables. We also examined cancer as part of our analysis. Participants who were missing serum Klotho and apoB were excluded, leaving us with 6647 participants with complete data (as depicted in Fig. 1). All NHANES research participants from 2007 to 2016 provided informed consent and obtained approval from the Research Ethics Review Committee of the National Health Statistics Center.

Covariates
The demographic variables included age, Gender (male or female), Race (Mexican American, other Hispanic, non-Hispanic White, non-Hispanic Black, or Other Race), Ratio of family income to poverty, and Education level (High School Grad and Less Than, or above). Age is a continuous variable in the demographic data. Then, age was categorized into the following 3 groups: <=40 years old, 41 to 60 years old, and >60 years old. Less Than 9th Grade, 9th to 11th Grade (Includes 12th grade with no diploma), and High School Grad/GED or Equivalent were classified as High School Grad and Less Than group. Some College or AA degrees and College graduates or above were classified as high school grad above group. Therefore, education level was classified as High School Grad and Less Than and above.
The questionnaire variables included Hypertension (No or Yes), High cholesterol level (No or Yes), Diabetes (No or Yes), Drinking (No or Yes), Smoking (No or Yes), and Cancer (No or Yes). The group with a drinking frequency of 0 was classified as the non-drinking group, and the group of people with a drinking frequency >0 was classified as the drinking group. Daily and occasional smokers were classified as smoker and never smokers as nonsmokers. Therefore, Smoking status was classified as smokers and never smokers.

Data collection
Trained interviewers conducted a questionnaire survey to collect data on Gender, Age, Race, Education level, Ratio of family income to poverty, Hypertension, High cholesterol level, BMI, Diabetes, Drinking, Smoking, and Cancer. Participants also underwent physical examinations and blood samples were collected to measure apoB (g/L) and Klotho (pg/mL) levels. Details of the NHANES laboratory methodology for Klotho determination are available at: https://wwwn.cdc.gov/Nchs/Nhanes/2007-2008/ SSKL_E.htm. Details of the NHANES laboratory methodology for apoB determination are available at: https://wwwn.cdc.gov/ Nchs/Nhanes/2007-2008/APOB_E.htm. Covariates can be found at the following URL: www.cdc.gov/nchs/nhanes/.

Data analysis
To study the associations of participant characteristics, we utilized weighted linear regression and the Chi-square test where appropriate. Firstly, we conducted a weighted univariate analysis to examine the relationship between Klotho and the covariates. Next, we performed weighted multifactor analyses while    Additionally, we conducted smooth curve fitting using apoB (g/L) as the abscissa and Klotho (pg/mL) level as the ordinate to confirm the relationship between apoB and serum Klotho. All statistical analyses were carried out using Empowerstats (https://www.empowerstats.net/cn/) and R software, and all estimates were weighted using the appropriate NHANES sample weight. We utilized weighted models to account for the oversampling of minorities, as suggested by the Centers for Disease Control and Prevention, to ensure a final unbiased and accurate estimate of effects for the population. Findings were deemed statistically significant at a P value of <.05.

Results
The table shows 14 study variables, which are composed of Serum Klotho, ApoB, Gender, Age, Race, Education level, Ratio    of family income to poverty, Hypertension, High cholesterol level, BMI, Diabetes, Drinking, Smoking, and Cancer. A total of 6647 participants were selected, with apoB as the independent variable, and they were divided into 3 groups for research (T1 group N = 2147; T2 group = 2195; T3 group = 2305) ( Table 1).
In the single-factor regression analysis, 13 variables of the study are displayed by serum Klotho. As can be seen from Table 2, in addition to apoB and Klotho having a certain correlation, gender, age, race, and high cholesterol levels are also related to Klotho. Table 3  It can be seen that after adjusting the relevant variables, apoB was linearly correlated with Klotho, and Klotho decreased with the increase of apoB (Figs. 2 and 3).

The result shown in
In the hypertensive population, Klotho increases first and then decreases with the increase of apoB. In the non-hypertensive population, Klotho showed a downward trend with increasing apoB, and then the downward trend tended to slow down. The results were obtained after adjusting for Gender, Age, Race, Education level, Ratio of family income to poverty, High cholesterol level, BMI, Diabetes, Drinking, Smoking, and Cancer (Fig. 4).
In alcohol drinkers, apoB was linearly correlated with Klotho, and Klotho decreased with the increase of apoB. In the non-alcohol drinkers, Klotho increases first and then decreases with the increase of apoB. The results were obtained after adjusting for Gender, Age, Race, Education level, Ratio of family income to poverty, Hypertension, High cholesterol level, BMI, Diabetes, Smoking, and Cancer (Fig. 5).
In the smoker population, apoB was linearly correlated with Klotho, and Klotho decreased with the increase of apoB. In the

Discussion
According to this study, there is a negative linear correlation between apoB and Klotho in the general population, as shown in Figures 2 and 3. Specifically, an increase in apoB is associated with a decrease in Klotho levels. This relationship holds true even when several cardiovascular risk factors are present, such as drinking, smoking, and high cholesterol level (Figs. 5-7). However, the relationship between apoB and Klotho in hypertensive and diabetic populations follows a different pattern (Figs. 4 and 8). In these populations, Klotho initially increases with an increase in apoB before decreasing, as seen in Figures 4  and 8. Importantly, the correlation between apoB and Klotho remains significant even after adjusting for potential confounders. This study is supported by 2 key theoretical concepts: the high correlation between apoB and LDL-C, and the role of apoB as a potential risk factor for CVD. [20] As such, elevated levels of apoB may serve as an important predictor of CVD risk, particularly in individuals with normal or low LDL-C levels. Therefore, further exploration of the relationship between apoB and Klotho may be a useful tool for identifying potential CVD patients within this population.
To the best of our knowledge, this study is the first to investigate the association between apoB concentration and serum Klotho concentration in the general population of the United States. Our findings suggest that specific CVD risk factors may be linked to lower Klotho levels, and controlling modifiable factors may help prevent or alleviate the decline of Klotho. The expression level of Klotho protein is also related to aging, and Kuro-o found that mice lacking Klotho suffer from premature aging syndrome. [23] Previous evidence has shown that Klotho deficiency and CKD cause aging, CVD, and bone disease. [24] Furthermore, there is a strong association between aging and the incidence of several age-related chronic diseases, such as diabetes and inflammatory bowel disease. [25,26] Thus, an unexpected discovery was made that apoB may be related to aging.
Several studies are currently investigating the potential association between the Klotho protein and dyslipidemia. Previous research has demonstrated that decreased serum Klotho levels and Klotho gene expression in the coronary artery wall are linked to the severity of coronary heart disease. [27] Animal studies have also indicated that Klotho protein could potentially promote adipocyte differentiation and play an anti-atherosclerotic role, decreasing the risk of CVD. [28] Recent studies have found a significant correlation between Klotho and blood lipids such as total cholesterol and triglyceride. [29] However, to date, there has been no investigation into the relationship between apoB and Klotho. In this study, we examined the correlation between apoB and Klotho while considering a range of potential confounding factors, including gender, age, race, education level, ratio of family income to poverty, hypertension, high cholesterol level, BMI, diabetes, drinking, smoking, and cancer. We utilized a linear regression model and smooth curve fitting to assess the association between apoB concentration and serum Klotho concentration and discovered a negative correlation between the 2.
This study stands out for being the pioneer in using public data from NHANES to explore the correlation between serum Klotho and the potential risk factor for CVD, apoB. Despite its strengths, it is important to acknowledge that this study has some limitations. Firstly, due to the cross-sectional nature of the NHANES database, we cannot infer any causal relationship between apoB and Klotho in humans. Secondly, there is a possibility of unknown confounding variables that could affect serum Klotho and apoB levels and were not considered in the study.

Conclusions
The Klotho protein is believed to play a critical role in regulating the onset and progression of CVD and other systemic illnesses. Investigating the function of Klotho and its impact on blood biochemical markers can be highly valuable in uncovering its role in CVD risk factors, thereby guiding clinical and prospective scientific research. Gene therapy targeting the Klotho gene may also hold promise in delaying aging and enhancing the quality of life of patients. With apoB high correlation with LDL-C and its potential as a CVD risk factor, identifying individuals at risk of CVD among the population with normal or low LDL-C levels by exploring the relationship between apoB and Klotho could be advantageous. A low serum Klotho level is associated with apoB, indicating that the soluble Klotho level may serve as a potential therapeutic marker for CVD prevention. However, more research is needed to clarify the Klotho gene precise control mechanism and the relationship between the specific transfer signal of the Klotho gene protein and cardiovascular risk factors.